DNA sequencing using hydrogel beads

Abstract

Systems, methods, and compositions provided herein relate to preparation of beads encapsulating long DNA fragments for high-throughput spatial indexing. Some embodiments include preparation of nucleic acid libraries within the bead, wherein the bead includes pores that allow diffusion of reagents while retaining genetic material.

What is claimed is: 
     
       1. A hydrogel bead for performing DNA reactions, comprising:
 a hydrogel polymer precursor, wherein the hydrogel polymer precursor is polyacrylamide; 
 a reversible polymer crosslinker that degrades in the presence of a reducing agent, wherein the reversible polymer crosslinker is N,N′-bis(acryloyl)cystamine (BACy); and 
 DNA disposed within the hydrogel bead, wherein the bead comprises pores that allow diffusion of a reagent through the bead while retaining the DNA. 
 
     
     
       2. The bead of  claim 1 , wherein the bead has a diameter of about 50 μm to about 150 μm. 
     
     
       3. The bead of  claim 1 , wherein the DNA is a long DNA molecule of 50,000 base pairs or greater. 
     
     
       4. The bead of  claim 1 , wherein the reagent comprises enzymes, chemicals, and primers having a size of less than 50 base pairs. 
     
     
       5. The bead of  claim 1 , wherein the reagent comprises lysozyme, proteinase K, random hexamers, polymerase (Φ29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (Tn5), primers (P5 and P7 adaptor sequences), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers, or bivalent cations. 
     
     
       6. A flow cell device for performing DNA sequencing, comprising:
 a solid support comprising a surface having a degradable hydrogel bead of  claim 1  encapsulating DNA deposited thereon, wherein the degradable hydrogel bead comprises pores that are sized to allow diffusion of a reagent through the hydrogel, but are too small to allow DNA to traverse the pores. 
 
     
     
       7. The flow cell device of  claim 6 , wherein the solid support is functionalized with a surface polymer. 
     
     
       8. The flow cell device of  claim 7 , wherein the surface polymer is poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) or silane free acrylamide (SFA). 
     
     
       9. The flow cell device of  claim 6 , wherein the flow cell comprises a patterned surface. 
     
     
       10. The flow cell device of  claim 9 , wherein the patterned surface comprises wells. 
     
     
       11. The flow cell device of  claim 10 , wherein the wells are from about 10 μm to about 50 μm in diameter, such as 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, or 50 μm in diameter, or within a range defined by any two of the aforementioned values, and wherein the wells are about 0.5 μm to about 1 μm in depth, such as 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, or 1 μm in depth, or within a range defined by any two of the aforementioned values. 
     
     
       12. The flow cell device of  claim 9 , wherein the wells are comprised of hydrophobic material. 
     
     
       13. The flow cell device of  claim 12 , wherein the hydrophobic material comprises an amorphous fluoropolymer, such as CYTOP, Fluoropel®, or Teflon®. 
     
     
       14. The flow cell device of  claim 6 , wherein the hydrogel bead has a diameter of about 50 pm to about 150 pm. 
     
     
       15. The flow cell device of  claim 6 , wherein the DNA is a long DNA molecule of 50,000 base pairs or greater. 
     
     
       16. The flow cell device of  claim 6 , wherein the reagent comprises enzymes, chemicals, and primers having a size of less than 50 base pairs. 
     
     
       17. The flow cell device of  claim 6 , wherein the reagent comprises lysozyme, proteinase K, random hexamers, polymerase (Φ29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (Tn5), primers (P5 and P7 adaptor sequences), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers, or divalent cations. 
     
     
       18. A system for DNA sequencing, comprising:
 a stage configured to hold a flow cell device of  claim 6 ; 
 a flow cell device of  claim 6 ; and 
 a detector for obtaining sequencing data. 
 
     
     
       19. A method of sequencing DNA comprising:
 obtaining a bead encapsulating DNA of  claim 1 ; 
 amplifying DNA encapsulated within the hydrogel; 
 performing a tagmentation reaction on the DNA encapsulated within the hydrogel; and 
 sequencing the DNA, 
 thereby generating a DNA library encapsulated within the hydrogel. 
 
     
     
       20. The method of  claim 19 , wherein the DNA is a long DNA molecule of 50,000 base pairs or greater. 
     
     
       21. The method of  claim 19 , further comprising performing a DNA amplification reaction on DNA encapsulated within the hydrogel prior to performing the tagmentation reaction. 
     
     
       22. The method of  claim 21 , wherein the DNA amplification reaction comprises multiple displacement amplification (MDA). 
     
     
       23. The method of  claim 19 , wherein the tagmentation reaction comprises contacting genetic material with a transposase mixture comprising adapter sequences and transposomes. 
     
     
       24. The method of  claim 19 , further comprising seeding the DNA library on a solid support. 
     
     
       25. The method of  claim 24 , wherein seeding comprises cleaving the hydrogel to release the DNA library from the hydrogel. 
     
     
       26. The method of  claim 25 , wherein the hydrogel is cleaved by contacting the hydrogel with a cleavage mix or by heating the hydrogel to about 90° C. to release the DNA library. 
     
     
       27. The method of  claim 26 , wherein the cleavage mix comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or tris(3-hydroxypropyl)phosphine (THP). 
     
     
       28. The method of  claim 24 , wherein the solid support is a flow cell device. 
     
     
       29. The bead of  claim 1 , wherein the reducing agent is dithioerythritol (DTE), dithiothreitol (DTT), 2-mercaptoethanol or 3-mercaptoethanol (BME), 2-mercaptoethanol, glutathione, thioglycolate, 2,3-dimercaptopropanol, tris(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THP), or P-[tri s(hydroxymethyl)phosphine] propionic acid (THPP).